Diabetes is a devastating disease of immense proportions. It is characterized by an impaired glucose metabolism that leads to, among other things an elevated blood glucose level (hyperglycemia) in diabetic patients. Type 1 diabetes is caused by autoimmune destruction of insulin-secreting β-cells within islets of Langerhans in the pancreas. Diabetes is classified into type 1, or insulin dependent diabetes mellitus (IDDM), which arises when a patient's β-cells cease producing insulin in their pancreatic glands, and type 2, or non-insulin dependent diabetes mellitus (NIDDM), which occurs in patients with an impaired insulin metabolism and β-cell malfunction. NIDDM usually takes decades to develop and is characterized sequentially by hyperinsulinemia, elevated triglycerides, high blood glucose and finally in late stages β cell fatigue, where insulin levels drop precipitously usually requiring insulin administration to the patient. In IDDM patients, the β-cells are selectively destroyed by an autoimmune process that involves lymphocyte infiltration. Early in the course of NIDDM, β-cell mass increases to meet the demand for more insulin. Loss of β-cell mass may then occur as NIDDM advances. β-Cells secrete insulin in response to changes in blood glucose concentration in highly regulated fashion and are responsible for achieving minute-to-minute regulation within physiological levels. Insulin deficiency results in prolonged hyperglycemia with serious long-term complications. Current treatments (e.g. insulin injections) do not provide tight regulation of blood glucose levels and thus do not alleviate the long-term complications of diabetes. Both naked and encapsulated islet transplantation are being explored as alternative treatments that can provide more physiological blood glucose level control. Islet transplantation is a promising method for restoring normoglycemia and alleviating the long-term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and will require a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that will ensure safety and in vivo efficacy.
The transplantation of immunoprotected insulin-secreting, glucose-responsive cells is a promising method for the long-term treatment of type 1 diabetes. A limitation that needs to be addressed before this methodology is implemented at a large-scale is cell availability. Human islets cannot be amplified in culture while retaining their differentiated secretory properties, so tissue from at least one human donor is needed for a single treatment of one recipient. The tissue availability limitation can be addressed by employing xenogeneic tissue (such as porcine islets) that is protected from the host's immune response. Immunoprotection can be achieved by enclosing the cells in a permselective membrane allowing passage of low molecular weight nutrients and metabolites, including insulin, but excluding larger antibodies and cytotoxic cells of the host. Most of the experimental work on encapsulated cell therapies has employed alginate as the encapsulating matrix. This methodology is particularly promising because it has the potential of restoring physiological regulation of blood glucose levels without the need for life long immunosupressive therapy. The feasibility of this approach in restoring normoglycemia has been demonstrated for diabetic animals and human with promising results (P. Soon-Shiong, et al., Lancet, 343, 950–1, 1994; R. P. Lanza, D. M. Kuhtreiber, et al., Transplant. Res., 28, 820, 1996; E. H. Liu, K. C. Herold, Trends Endocrinol. Metab. 11, 379–82, 2000; A. M. Shapiro, J. R. Lakey, et al., N. Engl. J. Med., 343, 230–238, 2000.).
Individuals at risk for developing IDDM can be identified by certain techniques. Those at risk for NIDDM are identifiable through family history and measurement of insulin resistance. However, little is known about the natural history of β-cell mass, turnover and cell lifetime, or the course of inflammation in diabetes. This is attributable to the highly heterogeneous nature of the pancreas, difficulties in its biopsy, and the low volume of β-cell mass (only 1–2% of the organ). Although insulin secretory capacity can be measured, it poorly reflects β-cell mass. There is therefore a substantial need for diagnostic methods that would enable (i) high-risk individuals to be monitored prior to onset of diabetes; (ii) diabetes patients to be monitored over the course of their disease to determine the exact stage of their disease; and (iii) also monitoring responses to therapy.
Current therapeutics for Type 1 diabetics are insulin or insulin mimetics, while most type 2 diabetic patients are treated either with agents that stimulate β-cell function or enhance the patient's tissue sensitivity towards insulin. Several classes of drugs are available for diabetes therapy. These include: insulin, or insulin mimetics; insulin sensitizers including (a) biguanides such as Metformin (b) retinoid-X-receptor (RXR) and peroxisome proliferator activated receptor (PPAR) agonists, such as the Thiazolidinedione (glitazone)and PPAR-γ agonists, e.g., Rosiglitazone and Troglitazone; (c) sulfonylureas (SU), such as Gliclazide, Glimepiride, Glipizide, Glyburide, Tolbutamide and Tolcyclamide; (d) amino acid and benzoic acid derivatives, such as Nateglinide and Repaglinide; (e) α-glucosidase inhibitors, such as Acarbose; (f) cholesterol lowering agents, such as (i) HMG-CoA reductase inhibitors, e.g., Lovastatin, and other statins), (ii) bile acid sequestrants, e.g., Cholestyramine (iii) nicotinic acid (iv) proliferator-activator receptor α-agonists, such as Benzafibrate, and Gemfibrozil, (v) cholesterol absorption inhibitors, e.g., β-sitosterol and (vi) acyl CoenzymeA:cholesterol acyltransferase inhibitors, e.g., Melinamide, and (g) Probucol.
Whilst continuous efforts are directed at developing new anti-diabetic agents, there is also a considerable need for the development of materials related to known therapeutic agents that may display improved bioavailability, functionality or reduced levels of undesirable effects. There is also a need for new diagnostic agents that can facilitate elucidation of the mechanism of insulin release or sensitization and the binding mechanism of the known anti-diabetic agents to their respective molecular receptors.
Fluorocarbon compounds and their formulations have numerous applications in medicine as therapeutic and diagnostic agents and as blood substitutes. Fluorine features a van der Waals radius (1.2A) similar to hydrogen (1.35A). Hydrogen replacement (with F) does therefore not cause significant conformational changes and fluorination can lead to increased lipophilicity, enhancing the bioavailability of many drugs. Fluorinated materials are often biologically inert and are generally expected to reduce side-effect profiles of drugs. The carbon-fluorine bond strength (460 kJ/mol in CH3F) exceeds that of equivalent C—H bonds. Perfluorocarbons (PFCs) display high chemical and biological inertness and a capacity to dissolve considerable amounts of gases, particularly oxygen, carbon dioxide and air per unit volume. PFCs can dissolve about a 50% volume of oxygen at 37° C. under a pure oxygen atmosphere. Fluorocarbon compositions can be used for wound treatment, as described in U.S. Pat. No. 4,366,169. Fluorocarbon formulations are also useful in diagnostic procedures, for example as contrast agents (Riess, J. G., Hemocompatible Materials and Devices: Prospectives Towards the 21st Century, Technomics Publ. Co, Lancaster, Pa. USA, Chap 14 (1991); Vox Sanguinis, 61:225–239, 1991).
Nuclear magnetic resonance (NMR) techniques permit the assessment of biochemical, functional, and physiological information from patients. Magnetic resonance imaging (MRI) of tissue water can be used to measure perfusion and diffusion with submillimeter resolution. Magnetic resonance spectroscopy may be applied to the assessment of tissue metabolites that contain protons, phosphorus, fluorine, or other nuclei. The combination of imaging and spectroscopy technologies has lead to spectroscopic imaging techniques that are capable of mapping proton metabolites at resolutions as small as 0.25 cm3 (Zakian K L; Koutcher J A; Ballon D; Hricak H; Ling C C, Semin Radiat Oncol.; 11(1):3–15, 2001). In magnetic resonance angiography (MRA) contrast agents are used to image the arteries and veins for diagnosing cardiovascular disease and associated disorders.
Of particular interest is fluorine's diagnostic value in non-invasive imaging applications. Apolar oxygen imparts paramagnetic relaxation effects on 19F nuclei associated with spin-lattice relaxation rates (R1) and chemical shifts. This effect is proportional to the partial pressure of O2 (pO2). 19F NMR can therefore probe the oxygen environment of specific fluorinated species in cells and other biological structures.
Nöth et al. (Nöth U; Grohn P; Jork A; Zimmermann U; Haase A; Lutz, J., 19F-MRI in vivo determination of the partial oxygen pressure in perfluorocarbon-loaded alginate capsules implanted into the peritoneal cavity and different tissues, Magn. Reson. Med. 42(6):1039–47, 1999) employed perfluorocarbon-loaded alginate capsules in MRI experiments to assess the viability and metabolic activity of the encapsulated materials. Quantitative 19F-MRI was performed on perfluorocarbon-loaded alginate capsules implanted into rats, in order to determine in vivo the pO2 inside the capsules at these implantation sites. Fraker et al. reported recently a related method with perfluorotributylamine (C. Fraker, L. Invaeradi, M. Mares-Guia, C. Ricordi, PCT WO 00/40252, 2000).
Ideally, PFC imaging agents should combine the following features: non-toxic, biocompatible, chemically pure and stable, low vapor pressure, high fluorine content, reasonable cost and commercial availability. Additionally, they should meet several 19F-NMR criteria, including a maximum number of chemically equivalent fluorines resonating at one or only few frequencies, preferably from trifluoromethyl functions. Some of the other spectral criteria have been discussed in detail elsewhere (C. H. Sotak, P. S. Hees, H. N. Huang, M. H. Hung, C. G. Krespan, S. Raynolds, Magn. Reson. Med., 29, 188–195, 1993.). For MRI, it would furthermore be desirable to have control over the amount of magnetically responsive material for specific uses, and to employ temperature-responsive and pH-dependent imaging agents for special uses. These could have applications in MRI-based temperature monitoring for use in general hyperthermia treatment (see, e.g., S. L. Fossheim; K. A. ll'yasov, J. Hennig, A. Bjornerud, Acad. Radiol., 7(12),1107–15, 2000.) of tumors and for monitoring the efficacy of chemotherapy, respectively (see, e.g., N. Rhagunand, R. Martinez-Zagulan, S. H. Wright, R. J. Gilles, Biochem. Pharmacol., 57, 1047–1058, 1999; I. F Tannock, D. Rotin, Cancer Res., 49, 4373–4383, 1989.). Furthermore, water solubility would enhance the PFC functionality in many biomedical settings, as it would obviate the need for emulsifiers.
Although selected efforts have been directed at developing new fluorinated MRI probes, none are water soluble compounds [e.g., perfluoro-[15]-crown-5 ether)], and some are commercially unavailable [e.g., perfluoro-2,2,2′,2′-tetramethyl-4,4′-bis(1,3-dioxalane)]. It appears no attempts have so far focused on screening available PFCs from the thousands of commercial fluorinated products in order to identify potentially more suitable MRI probes for biomedical uses. It seems furthermore that no studies have attempted to establish structure activity relations (SARs) of related PFCs for MRI purposes. Noteworthy is also the fact that all PFCs examined to date have molecular weights under 1,000, typically between 400–600 Da. This is partly a reflection of the specific requirements for blood substitution agents, but also due to the widely held belief that higher molecular weight or polymeric fluorinated agents would not be detectable by 19F-NMR due to anticipated excessive line broadening, and would therefore be unsuitable. Thus, with the exception of the polymer-encapsulated PFCs noted above, this important class of materials had so far been excluded from consideration.
Paramagnetic ions, such as gadolinium (Gd3+) decrease the T1 of water protons in their vicinity, thereby providing enhanced contrast. Gadolinium's long electron relaxation time and high magnetic moment make it a highly efficient T1 perturbant. Since uncomplexed gadolinium is very toxic, gadolinium chelate probes, such as gadolinium diethylenetriamine pentaacetic acid (GdDTPA Mw 570 Da), albumin-GdDTPA (Gadomer-17, Mw 35 or 65 kDa), have been employed extensively in MRI of tumors and other diseased organs and tissues. Several other developmental chelators have also been reported, including dual-labeled agents, oligonucleotide-derived, dextran-derived GdDTPA, and TAT and other peptide-derived chelators. However, presently approved MRI contrast agents are either not tissue specific, e.g., GdDTPA, or target only normal tissue, which limits their utility in diagnosis of metastases or neoplasia. MRI studies with GdDTPA, for instance, do not correlate with the angiogenic factor or the vascular endothelial growth factor (VEGF). Attempts have also been made to overcome the low relaxivities of small Gd-DTPA chelates by preparing polymer conjugates of Gd(DTPA)(2−) [see e.g., MRA. Duarte M. G.; Gil M. H.; Peters J. A.; Colet J. M.; Elst L. Vander; Muller R. N.; Geraldes C. F. G. C., Bioconjug. Chem., 21, 170–177, 2001.]. However, the relaxivity of these polymer conjugates was only slightly improved and they were also cleared very quickly from the blood of rats, indicating that they are of limited value as blood pool contrast agents for MRI.
Annexin V is a human protein (Mw 36,000) with high affinity for cells or platelet membranes that, following apoptosis (programmed cell death), have redistributed phosphatidylserine (PS) functions from internal to external membrane surfaces (see e.g., Verhoven et al. [B. Verhoven, R. A. Schlege, P. Willamson, J. Exp. Med., 182, 1597–1601, 1995] and Tait et al. [J. F. Tait, D. Gibson, J. Lab. Clin. Med., 123, 741–748, 1994.]). Apoptosis is an integral part of the aging and development of the central nervous system (CNS) and is linked to the pathogenesis of autoimmune and neurodegenerative diseases, cerebral and micordial ischemia, vasogenic edema, viral infections, inflammatory demyelinating diseases, organ and bone marrow transplant rejection, tumor response to chemotherapy and radiotherapy, and trauma [see e.g., H. Steller, Science, 267, 1445–1449, 1995; S. M. de la Monte, Y. K. Sohn, N. Ganju, J. R. Wands, Lab Invest., 158, 1001–1009, 1998.]. Among the neurodegenerative diseases linked to apoptotic events are Alzheimer's disease, Pick's disease, Parkinson's Disease, progressive supranuclear palsy, amyotrophic lateral sclerosis, and diffuse Lewy Body disease. These diseases are believed to share common neurodegenerative mechanisms, but maintain distinct clinical and pathological profiles due to atrophy and cell loss in specific regions of the CNS.
In view of the ubiquitous role of apoptosis in a broad range of disorders, a probe that could identify and quantify cell death in vivo would be of substantial benefit. The study of CNS neuron apoptosis could be a valuable tool for screening more effective drugs in the treatment of dementia associated with Alzheimer's and other diseases. One of the past hurdles in the development of potential therapies has been the general lack of relevant in vitro and in vivo diagnostic methodologies to assess the potential of new therapeutic compounds. Annexin's affinity for PS has been exploited to study in animals and humans hepatic apoptosis, chemotherapy, allograft rejection, and thrombosis, using radioisotope-labeled annexin [see e.g., Blankenberg et al., Proc. Natl. Acad. Sci. USA, 95, 6349–6354, 1998; Ohtsuki K; Akashi K; Aoka Y; Blankenberg F. G.; Kopiwoda S; Tait J. F.; Strauss H. W. Eur. J. Nucl. Med., 26, 1251–8, 1999. Blankenberg F. G.; Katsikis P. D.; Tait J. F.; Davis R. E.; Naumovski L.; Ohtsuki K.; Kopiwoda S.; Abrams M. J.; Strauss H. W., J. Nucl. Med., 40, 184–91, 1999. J. R. Stratton, et al., Circulation, 92, 3113–3121, 1995.]. Another PS binding protein, the C2 domain of synaptotagmin I, was conjugated to superparamagnetic iron oxide (SPIO) nanoparticles and used in MRI to detect apoptotic cells. Zhou, et al, Nature Medicine, Vol. 7, No.: 11, November 2001.
Whilst much can be achieved with currently available imaging and contrast agents, there are still unmet needs for novel diagnostic agents, particularly for those exploiting biological specificity. Imaging agents suitable for targeting metastases or neoplasia would substantially enhance the MRI sensitivity and utility for tumor detection and prevention. Similarly, imaging agents suitable for targeting receptors involved in insulin production and utilization would substantially enhance our understanding of the diabetes disease process and the function of anti-diabetic drugs. Although selected efforts have been directed at developing such new probes, a broader investigation of these agents is urgently needed. Similarly, new imaging probes are needed as noninvasive means to detect and image cells, tissues and organs undergoing apoptosis.